ABSTRACTDynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

fig7: Homotypic cell aggregation promotes LFA-1 macro- and microclustering at the cell–cell contact interface. (A) K562 transfectants expressing wild-type αLβ2 in 1.5-ml microfuge tubes (106 cells in 100 μl L15 medium + 2 mg/ml glucose) were centrifuged at 200 g for 30 s and incubated in the absence (control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D for 1 h at 37°C, transferred to cell culture dishes, and imaged by phase-contrast microscopy using a 10× objective in the original experiment. (B) Transient αL-mCFP/β2 and αL-mYFP/β2 K562 transfectants were treated exactly as in A, and inter-heterodimer FRET was measured for homotypically adherent cells. DIC (top), CFP fluorescence after YFP bleaching (middle), and pixel-by-pixel FRET efficiency (from 0 [black] to 0.2 [red]) (bottom) are shown. (C and D) K562 transfectants stably expressing αL-mCFP/β2-mYFP and the talin head domain (Kim et al., 2003) were gently removed from culture flasks and either directly plated on cover glasses and imaged with YFP fluorescence (C) or micropipetted 5–10 times to obtain single suspensions, plated, and incubated for 10 min before imaging YFP fluorescence (D). (E) Aggregates of K562 transfectants expressing αL-mCFP/β2-mYFP and the talin head domain were dissociated as in D and immediately subjected to time-lapse fluorescence imaging at 37οC. (F) To observe the formation of macroclusters, cells treated as in D were allowed to reestablish homotypic cell–cell contacts during time-lapse fluorescence imaging. Images are from representative experiments. (See also Video 2 and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1).

Mentions:
Many cell types, including K562 cells, and all leukocytes that express LFA-1 also express one or more of its ligands, the ICAMs, facilitating formation of aggregates of homotypically adherent cells (Springer, 1990; de Fougerolles and Springer, 1992). In all experiments described above, we avoided homotypic adhesion by activating cells at low density. Furthermore, cells were vigorously resuspended, allowed to settle, and incubated at 37°C for 10 min before fluorescence measurements to ensure that even if some preexisting homotypic adhesion had occurred, that the distribution of LFA-1 had equilibrated for 10 min after the cells were monodisperse. Under these conditions on ICAM-1 substrates, no homotypic adhesion occurred between neighboring K562 transfectants, as confirmed by the lack of redistribution of LFA-1 (Fig. 6 C, middle). However, when K562 transfectants were incubated at high cell densities with Mn2+, PMA, or cytochalasin D under conditions similar to those previously reported to lead to macroclustering of LFA-1, significant homotypic adhesion occurred (Fig. 7 A). Furthermore, marked macroclustering of LFA-1 was found at sites of cell–cell contact between homotypically adherent cells, whether stimulated with Mn2+, PMA, or cytochalasin D (Fig. 7 B). Moreover, LFA-1 was present in microclusters at the cell–cell contact zone, as revealed by inter-heterodimer FRET (Fig. 7 B, bottom). Notably, LFA-1 present outside the region of cell–cell contact was not microclustered.

fig7: Homotypic cell aggregation promotes LFA-1 macro- and microclustering at the cell–cell contact interface. (A) K562 transfectants expressing wild-type αLβ2 in 1.5-ml microfuge tubes (106 cells in 100 μl L15 medium + 2 mg/ml glucose) were centrifuged at 200 g for 30 s and incubated in the absence (control) or presence of 1 mM Mn2+, 1 μM PMA, or 1 μM cytochalasin D for 1 h at 37°C, transferred to cell culture dishes, and imaged by phase-contrast microscopy using a 10× objective in the original experiment. (B) Transient αL-mCFP/β2 and αL-mYFP/β2 K562 transfectants were treated exactly as in A, and inter-heterodimer FRET was measured for homotypically adherent cells. DIC (top), CFP fluorescence after YFP bleaching (middle), and pixel-by-pixel FRET efficiency (from 0 [black] to 0.2 [red]) (bottom) are shown. (C and D) K562 transfectants stably expressing αL-mCFP/β2-mYFP and the talin head domain (Kim et al., 2003) were gently removed from culture flasks and either directly plated on cover glasses and imaged with YFP fluorescence (C) or micropipetted 5–10 times to obtain single suspensions, plated, and incubated for 10 min before imaging YFP fluorescence (D). (E) Aggregates of K562 transfectants expressing αL-mCFP/β2-mYFP and the talin head domain were dissociated as in D and immediately subjected to time-lapse fluorescence imaging at 37οC. (F) To observe the formation of macroclusters, cells treated as in D were allowed to reestablish homotypic cell–cell contacts during time-lapse fluorescence imaging. Images are from representative experiments. (See also Video 2 and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200404160/DC1).

Mentions:
Many cell types, including K562 cells, and all leukocytes that express LFA-1 also express one or more of its ligands, the ICAMs, facilitating formation of aggregates of homotypically adherent cells (Springer, 1990; de Fougerolles and Springer, 1992). In all experiments described above, we avoided homotypic adhesion by activating cells at low density. Furthermore, cells were vigorously resuspended, allowed to settle, and incubated at 37°C for 10 min before fluorescence measurements to ensure that even if some preexisting homotypic adhesion had occurred, that the distribution of LFA-1 had equilibrated for 10 min after the cells were monodisperse. Under these conditions on ICAM-1 substrates, no homotypic adhesion occurred between neighboring K562 transfectants, as confirmed by the lack of redistribution of LFA-1 (Fig. 6 C, middle). However, when K562 transfectants were incubated at high cell densities with Mn2+, PMA, or cytochalasin D under conditions similar to those previously reported to lead to macroclustering of LFA-1, significant homotypic adhesion occurred (Fig. 7 A). Furthermore, marked macroclustering of LFA-1 was found at sites of cell–cell contact between homotypically adherent cells, whether stimulated with Mn2+, PMA, or cytochalasin D (Fig. 7 B). Moreover, LFA-1 was present in microclusters at the cell–cell contact zone, as revealed by inter-heterodimer FRET (Fig. 7 B, bottom). Notably, LFA-1 present outside the region of cell–cell contact was not microclustered.

Bottom Line:
Dynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration.Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand.Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering.

ABSTRACTDynamic regulation of integrin adhesiveness is required for immune cell-cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function-associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.